Aiming to address the seedling leakage issue caused by missing seedlings in fully automatic pepper tray seedling transplanters, the seedling separation process of the transplanter was analyzed and a dual-seedling cylinder independent drive system with empty-cell replenishment for seedling separation was proposed, aiming to minimize missed planting. An electric drive double group seedling cup common rail type separating seedling system was designed. The two sets of seedling cylinders operated on identical trajectories. While one set remained stationary to receive seedlings, the other set can continue performing seedling-dropping operations or execute empty-cell replenishment tasks, ensuring a continuous supply of seedlings to the planting device’s duckbill mechanism. An optical fiber identification sensor was selected for seedling absence detection, and a PLC-based automatic control system was established to acquire the sensor output signals in real time. Based on the determination of the empty-cell state derived from a binary threshold algorithm, the system triggered the actuator to initiate accelerated motion for empty-cell replenishment operations. With planting frequency, air supply pressure, and the status of the detection and compensation function (on/off) as experimental factors. The trial evaluated transplant failure rates and duckbill seedling reception rates of the planting device to identify optimal parameter combinations, followed by field validation. Results showed that under the combination of 120 plants per minute planting frequency and 0.4MPa air supply pressure with detection and compensation function was disabled, the average duckbill seedling reception rate was 86.46%. When the detection and compensation function was activated, the rate was increased to 98.27%, demonstrating a significant enhancement in seedling reception efficiency. This research can provide valuable insights for the development of seedling separation devices for chili pepper tray seedlings.